9 December 2007. Could oxidative stress underlie the pathology of schizophrenia? A paper in the December 7 issue of Science reports that reactive oxygen species mediate some of the effects of the NMDA receptor antagonist ketamine on inhibitory interneurons. Researchers led by Margarita Behrens and Laura Dugan at the University of San Diego, La Jolla, California, demonstrate that activation of NADPH oxidase, an enzyme that generates the toxic reactive oxygen species superoxide, is crucial for ketamine-induced disruption of parvalbumin-expressing interneurons. These inhibitory neurons play a crucial role in regulating excitatory neural networks in the brain, and there is evidence that their activity is compromised in schizophrenia patients. As ketamine and other NMDA-type glutamate receptor blockers induce psychotic behaviors indistinguishable from those seen in schizophrenia patients, the finding raises the possibility that in schizophrenia, like many other nervous system disorders, neurons may be particularly susceptible to oxidative stress.

Linking neurotransmitter hypotheses of schizophrenia
Fast-spiking interneurons expressing the calcium-binding protein parvalbumin (PV) modulate cortical networks via their release of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter. Postmortem analysis shows that this neuronal phenotype is depleted in the cortex of patients with schizophrenia (see, for example, Beasley et al., 2002; Hashimoto et al., 2003). There is evidence that these particular interneurons have not been lost, but are merely producing less parvalbumin, along with producing less GAD67, a GABA-synthesizing enzyme that is also depleted in schizophrenia (see SRF related news story).

Linking these GABAergic phenomena with glutamatergic signaling via the NMDA receptor, Behrens and colleagues have previously shown that ketamine exposure also depletes interneurons in culture of parvalbumin and GAD67 (Kinney at al., 2006). This suggests that blocking NMDA signaling in GABAergic neurons causes a loss in GABA transmission, which in turn frees glutamatergic neurons from inhibitory innervation and leads to heightened glutamate release. By a hypothetical feedback mechanism, cortical neuronal networks would respond to the excess glutamatergic signaling by reducing the GABAergic signaling through parvalbumin expressing interneurons.

In support of this model, Behrens and colleagues now report that adding the GABA agonist muscimol to mouse neuronal cultures treated with ketamine—in effect, fooling the system into thinking that the GABAergic interneurons are performing normally—protects GABAergic neurons from parvalbumin and GAD67 loss.

Oxidative stress as an underlying mechanism?
But what is it about NMDA antagonists that could cause loss of PV and GABA in the first place? Researchers have recognized that NMDA antagonists cause an increase in reactive oxygen species both in vivo and in vitro (Xia et al., 2002; Zuo et al., 2007), though it is not clear why. But Behrens and colleagues, noting recent reports that NADPH oxidase is found in the brain, wondered if this enzyme may be involved. Recent evidence suggests that the enzyme may have unappreciated roles in cellular communication, but its primary role appears to be generation of highly toxic superoxide for the destruction of engulfed bacteria in phagocytes.

Several lines of evidence suggest that Behrens and colleagues may have identified a pathway crucial to the loss of parvalbumin and GAD67 expression in GABAergic interneurons. They report that prolonged exposure to low levels of ketamine drives superoxide production in primary neuronal cultures, and that both a superoxide scavenger (C3) and an NADPH oxidase inhibitor (apocynin) protect the PV/GAD67 phenotype from ketamine.

In the bottom left panel of the figure above (figure 4B from the article), ketamine produces a red cloud of superoxide in mouse prelimbic cortex, with an accompanying loss of parvalbumin reactivity (green). But pretreatment to reduce superoxide production preserves parvalbumin reactivity as seen in the panels on the right. [Image credit: Behrens et al., Science. 2007 December 7;318:1645-1647. Reprinted with permission from AAAS.]

More significant, perhaps, is that pretreatment with both the scavenger and inhibitor also prevent the effects of ketamine in live animals. Analysis of mouse prefrontal cortex, an area of the brain thought to be particularly important for the psychopathology of schizophrenia, showed that ketamine induces a widespread increase in superoxide, which can be abrogated by C3 or apocynin. The effects extend to the PV-positive interneurons, which retain their parvalbumin and GAD67 when animals are prophylactically protected.

All told, the authors hypothesize that, "NADPH oxidase may be a contributor to oxidative mechanisms involved not only in the psychotomimetic effects of NMDA-R antagonists, but also in schizophrenia and other processes involving increased oxidative stress in the brain.” The work also contributes to the ongoing debate about the induction of schizophrenia-like symptoms in healthy people who take NMDAR antagonists “recreationally” (see comment from John Krystal below).

It is also worth noting that other researchers have recently made a connection between schizophrenia and oxidative stress, namely, a genetic polymorphism in a gene needed for synthesis of the antioxidant peptide glutathione (see related SRF news). It could be that oxidative stress may play a much greater role in the disease than previously anticipated.—Tom Fagan.

The paper by Behrens and colleagues provides exciting new...
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The paper by Behrens and colleagues provides exciting new data to suggest that NADPH oxidase plays an important role in the impact of the NMDA receptor antagonist, ketamine, upon parvalbumin-containing (PVC) fast-spiking GABA interneurons. The authors show that ketamine causes an activation of NADPH oxidase, resulting in increases in superoxide production. The elevation in free radicals, presumably toxic to these neurons, is associated with reduction in the expression of parvalbumin and GAD67. These effects of ketamine could be prevented by inhibition of NADPH oxidase.

These data were interpreted by the authors to help explain the schizophrenia-like effects of ketamine in healthy humans. I think that these data provide important insights into the impact of reductions in NMDA receptor function, and they may be relevant to schizophrenia. First, the data amplify the implications of the work of Kinney, Cunningham, and others who have shown that PVC interneurons express the NR2A subunit of the NMDA receptor and that deficits in NMDA receptor function may contribute to reduced GAD expression by these neurons. Since PVC deficits in GAD expression have been described in postmortem cortical tissue from people diagnosed with schizophrenia, the current data suggest that some of these findings may be attributable to activation of NADPH oxidase. It would be interesting to know whether there is an interaction between this consequence of deficits in NMDA receptor function, a feature associated with schizophrenia, and reductions in the cortical levels of glutathione, also associated with this disorder. Glutathione is a free radical scavenger. In other words, the emergence of GABA neuronal deficits may be an unfortunate consequence of the convergence of a disturbance in glutamatergic neurotransmission and a heritable abnormality in neural metabolism. These data highlight the potential importance of some very preliminary new data that suggest that N-acetyl-cysteine (NAC) may augment antipsychotic effects in treating schizophrenia. NAC raises intracellular glutathione and might be a treatment that targets the cellular process described by Behrens and colleagues.

The Behrens paper also highlights the importance of research studies exploring ketamine effects from a systems and cognitive neuroscience perspective. For example, it does not explain why ketamine effects produce symptoms and cognitive impairments associated with schizophrenia. It is likely that the work of scientists including H. Grunze, R. Greene, B. Moghaddam, R. Dingeldine, M. Cunningham, and others is important to consider. These investigators have shown that NMDA receptor antagonists reduce the recruitment of PVC interneurons in feed-forward inhibition pathways resulting in increased glutamatergic output. When NMDA receptors are blocked, the activity of these neurons produces dysfunctional effects, in that neural activity seems chaotic and the organized oscillatory activity of networks is disrupted. These disturbances in network function are paralleled by abnormal behaviors and cognitive impairments in animals and "schizophrenia-like" symptoms and cognitive deficits in humans. One potential solution to this problem would be to reduce glutamate release, a paradoxical suggestion for a disorder commonly thought of as "hypoglutamatergic" based on loss of cortical connectivity. Yet, in animals and humans, drugs that reduce glutamate release (lamotrigine, group II metabotropic glutamate receptor agonists) reduce the physiologic and behavioral effects of NMDA glutamate receptor antagonists. Further, there are now some encouraging clinical data that lamotrigine and, particularly, group II mGluR agonists, might have clinical efficacy in treating schizophrenia.

Overall, we seem to be working in a period where a wide variety of data from many sources is rapidly converging to capitalize on the insight that NMDA receptor antagonists, when administered to healthy people, transiently produce effects that resemble schizophrenia.

The article by Behrens and colleagues provides evidence for a mechanistic link between NADPH oxidase and disruption of normal protein expression in some interneurons following the drug ketamine. Data presented demonstrate that addition of an NADPH oxidase inhibitor, given in the animal’s drinking water, blocked the effects of ketamine on a specific class of interneurons that contains parvalbumin. Several lines of research suggest that this population of cells is disrupted in schizophrenia, and that reductions of NMDA-type glutamate receptor activity may lead to that impairment. The important iterative advance in the current study links the reduction in NMDA receptor-mediated glutamate transmission to a specific intracellular mechanism and molecular pathway. Furthermore, the authors demonstrate that they can effectively block the cellular changes by inhibiting that pathway, suggesting a novel therapeutic target.

This leads to two major questions: 1) Could NADPH oxidase inhibitors, or similar mechanisms be used to avert the onset of schizophrenia if administered during a prodromal period? 2) Is the process of reduced parvalbumin expression reversible? Some studies have shown that drugs like ketamine, which reduce activity at NMDA receptors, actually lead to cell death, suggesting that only prevention would be possible. Alternatively, there is evidence that the parvalbumin-containing cells in schizophrenia may not be dead and gone, but rather have impaired function and loss of this particular protein. In this latter scenario, it is possible that the effects of the illness could be reversible. Given that ketamine also causes a variety of functional changes in animals, including electrical brain activity and behavior, the current work lays the groundwork for future studies to determine if co-administration of NADPH oxidase inhibitors can block the functional consequences of ketamine and, by extension, reduce NMDA receptor activity in general.

The study by Behrens and colleagues is an excellent illustration of how breaking with traditional paradigms can lead to identification of novel potential targets for intervention in schizophrenia. As detailed on the pages of Schizophrenia Research Forum (e.g., Interview with D. Lewis) and the cited articles from F. Benes, one of the most consistent findings in schizophrenia is the downregulation of PV and GAD67 expression in PV+ GABAergic interneurons. Dysfunction of these neurons, in turn, may be responsible for frontal neurocognitive and dopaminergic deficits. The underlying cause of the GABAergic interneuron changes, however, has only intermittently been investigated.

One of the leading potential mechanisms underlying reduced PV and GAD67 expression in brain in schizophrenia has always been NMDA dysfunction, given the strong expression of NMDA receptors on GABA interneurons, as described by Behrens and colleagues, and the well-known ability of NMDA antagonists to induce both symptoms and neurocognitive deficits closely resembling those of schizophrenia. Last year, Kinney and colleagues demonstrated that exposure to the NMDA antagonist ketamine reduced PV and GAD67 expression in GABAergic interneurons in vitro (Kinney et al., 2006). The present study builds upon this finding and demonstrates a similar phenomenon in vivo. Moreover, it builds upon this finding to demonstrate that these changes can be reversed by antagonists of NADPH oxidase, suggesting a potential target for intervention.

This study thus adds reduced GAD67 and PV expression in PV+ GABAergic interneurons to the long list of findings in schizophrenia that can be viewed as “downstream” of a more proximal deficit in NMDA-mediated neurotransmission, and supports interventions aimed specifically at frontal GABAergic interneurons, as well as more generally at reduced NMDA activity throughout brain. This preparation, moreover, may be appropriate to the testing of novel glutamatergic agents.

Behrens and colleagues' article, however, also leaves many questions unanswered. For example, loss of PV and GAD67 in schizophrenia is not confined to prefrontal cortex. It would be of interest to know, therefore, whether histological changes induced by ketamine are or are not confined to this region. As with all proposed new drug targets, it will also be important to know what other processes NADPH oxidase is involved with both inside and outside brain before proposing it too seriously as a drug target. It is one thing to reverse a specific deficit in a short-term treatment model, another to contemplate long-term treatment. At first glance, NADPH oxidase would seem to be a very general enzyme, which is being targeted to treat a very specific condition. Nevertheless, if NADPH oxidase activity can safely be blocked throughout the body long term, the present findings may point the way for new treatments for schizophrenia.

The role of reactive oxygen species in the pathogenesis of...
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The role of reactive oxygen species in the pathogenesis of schizophrenia is currently unclear. Several lines of evidence support a greater production of these reactive molecules in schizophrenia because of reduced levels of important buffers for superoxides, such as glutathione. Other research, however, suggests that antipsychotic drugs themselves increase the production of oxygen radicals. In this week’s issue of Science, Behrens and colleagues present data supporting the involvement of reactive oxygen species in the pathophysiology of schizophrenia. The authors have previously shown that administration of an NMDA receptor antagonist to primary cultures of cortical neurons results in the loss of GAD67 and parvalbumin (PV; a calcium-binding protein) from PV positive GABAergic interneurons (Kinney et al., 2006), similar to what has been observed in studies using postmortem tissue from patients with schizophrenia (e.g., Volk et al., 2000; Hashimoto et al., 2003). In this study, administration of the NMDA receptor antagonist ketamine was found to increase production of reactive molecules both in vitro (following bath application of the drug to cultured neurons) and in vivo (following two injections of the drug to mice). Moreover, inhibition of the enzyme NADPH oxidase prevented the reduction of both PV and GAD67 expression. The authors suggest that inhibition of NADPH oxidase may represent a novel treatment for both ketamine-induced psychosis and schizophrenia.

While the authors’ findings are undoubtedly exciting, some limitations of their approach need to be addressed before over-enthusiasm regarding NADPH oxidase inhibition as a treatment for schizophrenia is generated. Although the title advertises a “loss of phenotype of fast-spiking interneurons,” the reduction in PV and GAD67 expression from neurons that remain PV positive does not represent a loss of phenotype, and the ketamine-induced increase in superoxide production was not specific to interneurons (only 5-10 percent of primary cortical neuron cultures are PV positive, yet the effect was observed throughout sampled cells). Also, although their findings bear similarity to those observed in schizophrenia, there are notable differences. For instance, whereas the level of PV expression per cell is reduced in schizophrenia (Hashimoto et al., 2003), the level of GAD67 mRNA expression per cell does not differ between individuals with schizophrenia and controls; rather, it appears to be a reduction in the density of neurons that express the transcript. In contrast, Behrens and colleagues report a reduction in the expression per cell for both PV and GAD67. While this difference may simply be due to the fact that Behrens and colleagues examined levels of the proteins, the potential discrepancy should be recognized.

Perhaps the most important limitation to the work is the absence of a functional measure to determine whether the reduction in PV and GAD67 in cortical interneurons observed following ketamine administration results in any of the schizophrenia-associated endophenotypes which can be modeled in rodents. Animal models of schizophrenia employing developmental strategies have been very successful in this regard (reviewed in Carpenter and Koenig, in press), and it is unclear how functional outcomes from the acute pharmacological challenge in mature animals used in the present study might compare. Although the data as they stand are promising, they would be much more compelling if a functional deficit as a result of the treatment was observed and the authors could demonstrate that inhibition of NADPH oxidase prevented this deficit. Unfortunately, such a deficit is unlikely to be found following such a limited ketamine exposure. This is actually quite fortunate since ketamine is a popular general anesthetic in both human and veterinary medicine. Additionally, countless biomedical investigators routinely use ketamine as an anesthetic for survival surgeries; even in cases where the experimental design calls for multiple anesthetizations over the course of the study, no major functional disturbances in experimental animals have been reported. Our conclusion is that, while exposure to ketamine may induce features of neuropathology that bear some similarity to those observed in schizophrenia, the excitement about a treatment for ketamine-induced superoxide production should be tempered until it can be demonstrated that the treatment reverses a functional deficit that is relevant to schizophrenia.

For two decades, following the work by Benes and her colleagues, it has been increasingly apparent that there is a deficit in cortical GABAergic neurons in schizophrenia. Ten years ago we found that the parvalbumin (PV)-containing, but not calretinin-containing, subgroup of these neurons was selectively affected, and recently this specific deficit has been seen in animal models of the disease. Repeated administration of non-competitive NMDA receptor antagonists such as PCP, MK801, and ketamine can induce in rats some behaviors reminiscent of schizophrenia, as well as enduring deficits in PV expression.

Behrens and colleagues have identified some of the molecular mechanisms underlying this specific neurotoxicity of ketamine and, probably, other NMDA antagonists. That the effects of ketamine involve generation of reactive oxygen species (ROS) is not surprising, given the ubiquity of oxidative free radical production in neurotoxic processes. However, identifying the role of NADPH oxidase in producing ROS in response to ketamine, and demonstrating that this process determines the consequent toxic effects of ketamine on PV-containing and other neurons, are potentially important developments.

The importance of these findings to schizophrenia relies on the assumption that repeated administration of ketamine and, presumably, other NMDA antagonists not only models (some of) the pathophysiology of schizophrenia, it also mimics the process leading to this neuronal pathology. This is far from proven, although the NMDA receptor hypofunction hypothesis of Olney and Farber provides a useful model mechanism for this pathogenesis.

A useful proof of concept would be to move away from pharmacological approaches to other animal models of the disease. One such is the isolation rearing paradigm; in this model, induction of abnormal “schizophrenia-like” behaviors is also paralleled by a deficit in PV-containing neurons (Harte et al., 2007). A simple but very informative study here would be to determine whether inhibition of NADPH oxidase might protect against the development of these deficits. Of course, how the NMDA receptor-mediated deficits relate temporally to the natural history of schizophrenia is unclear; we do not know when the PV deficits occur in schizophrenia. There may be some hope for targeted treatment with, e.g., NADPH oxidase inhibitors if the neuronal pathology parallels a neurotoxic process that underlies the progressive cognitive disturbances as implied by Olney and Farber, but not if the PV deficits relate to an early and primary pathology of the disease.

The recent study by Behrens and colleagues provides in vitro evidence that blockade of NMDA receptors by ketamine leads to a selective reduction in PV and GAD67 that appears to be due to the toxic effects of superoxide anion arising subsequent to the activation of NADPH oxidase. Blockade of the sublethal, toxic effects of ketamine in neuronal culture is consistent with our report demonstrating that the apoptotic effect of phencyclidine (PCP) on cortical neurons in vivo also could be prevented by the superoxide dismutase mimetic, M40403 (Wang et al., 2003). Though seemingly non-specific, superoxide dismutase mimetics may prove to be useful in the treatment of ketamine or PCP-induced psychosis because of the relative sparseness of critical life-promoting processes that require superoxide anion. Perhaps more importantly, a better understanding of the mechanisms underlying ketamine-induced loss of PV/GAD67 may lead to novel treatment modalities for schizophrenia.

While the primary focus of the report by Behrens and colleagues is on PV-expressing GABAergic interneurons, Fig. 1 clearly demonstrates that ketamine also affects a large population of non-PV neurons. This is consistent with our recent in vivo experiments in developing rats demonstrating that PCP administration on PN7 induces apoptosis of cortical PV-containing interneurons as well as principal neurons in layers II-IV of the cortex (Wang et al., 2007). Early postnatal administration of PCP also results in neuronal apoptosis in the hippocampus, striatum, and thalamus (Wang and Johnson, 2007. Thus, it may be premature to focus solely on this population of interneurons.

In thinking about the mechanism underlying the selective loss of PV interneurons following PCP, it is important to note that PV is not yet expressed on PN7, which is when PCP was administered in our paradigm (Wang et al., 2007). (The loss of PV-containing interneurons was measured at PN56, well after the time of PV expression on about PN10.) Interestingly, interneurons expressing calretinin and calbindin at the time of PCP administration were spared. These neurons showed no colocalization with cellular markers of apoptosis (terminal dUTP nick-end labeling [TUNEL] of broken DNA or cleaved caspase-3), indicating that calretinin- and calbindin-containing neurons were protected from the toxic effect of PCP and survived into adulthood (Wang et al., 2007). The mechanism underlying this selectivity for cortical PV-containing interneurons is unknown, but as Behrens and colleagues suggest, it could be because these neurons are dependent on a relatively large glutamatergic input for survival. It is also possible that the differing calcium buffering capacities of these interneurons play a role in the selective neurotoxic effect of NMDA receptor blockade. That is, since calcium binding proteins could also act to buffer decreases in intracellular Ca2+ levels caused by ketamine-induced blockade of NMDA receptors, it is possible that the lack of PV in these vulnerable interneurons reduces the ability of these cells to adequately buffer the ketamine-induced decrease in intracellular calcium. This is consistent with the lack of effect on other interneurons that express the calcium binding proteins calretinin and calbindin at the time of PCP administration. This suggests NMDA receptor blockade could cause the deletion of PV neurons because of a specific effect at a critical stage of development. However, cleaved caspase-3 (a hallmark of apoptosis) showed no colocalization with BrdU, a specific marker of S-phase proliferation (Wang et al., 2007). These data suggest that the loss of PV-containing neurons in this paradigm was not due to an effect of PCP on proliferating neurons, but rather an effect on postmitotic neurons.

We have reported recently that PCP in cortical neuronal culture causes neuronal apoptosis by interfering with the Akt-GSK-3β cascade, which is necessary for neuronal survival during development (Lei et al., 2007). Moreover, increasing synaptic strength by various means such as increasing calcium current via activation of L-type calcium channels completely blocks PCP-induced cell death by increasing Akt phosphorylation. It would be of great interest to determine whether PV-containing interneurons respond in a similar fashion.

In order to fully appreciate the significance of ketamine-induced loss of PV-containing neurons, it will be necessary to carefully compare the in vivo dose-related effects of ketamine or PCP that are truly selective for PV/GAD67-containing interneurons to those cortically mediated behaviors that have relevance to schizophrenia.